Isolation and sequencing of coral-associated viruses
Viral particles were physically isolated prior to DNA extraction and shotgun cloning. Fragments of healthy and partially bleached D. strigosa
colonies were collected in triplicate. Bleaching corals were visually identified as those that had lost 40–60% of their normal pigmentation. Tissue was removed from coral skeletons using an airbrush. Tissue in each set of triplicate samples was pooled to create two metagenomic libraries, DsH and DsB. (‘Ds’ in the sample name represents the coral species, D. strigosa
, while ‘H’ and ‘B’ indicate the corals' healthy or bleaching condition.) Pooling was not necessary to obtain sufficient viral material. However, we chose to pool samples to reduce the sample-to-sample variation associated with metagenomic data sets (Angly et al., 2006
). Pooling samples also allowed us to examine a broader diversity of coral-associated viruses, though it concealed any variability between the individual corals collected. The raw coral tissue blastate was too viscous for processing, so power homogenization was used to liquefy it. Virus counts before and after homogenization showed no significant difference in VLP abundance (Table S1; P
= 0.88). Centrifugation in cesium chloride density gradients was used to separate VLPs from cellular debris. Removal of microbial and eukaryotic cells was confirmed with epifluorescence microscopy (). To ensure that all microbial DNA was removed, the viral fraction from the cesium chloride gradient was treated with chloroform to lyse mitochondria. DNase I was then used to degrade the exposed DNA (Breitbart and Rohwer, 2005
Fig. 1 Epifluorescent micrographs of samples before and after viral particle isolation. DsH = Diploria strigosa – Healthy, DsB = Diploria strigosa – Bleaching. Samples were stained with SYBR Gold nucleic acid stain and visualized under epifluorescence (more ...)
DNA was extracted from the isolated viruses and cloned using the Linker Amplified Shotgun Library (LASL) method. In total, 1580 sequences from the DsH library and 930 sequences from the DsB library were obtained. When compared with the GenBank non-redundant (NR) database using tblastx
, 44% of DsH sequences and 59% of DsB sequences had significant hits to known sequences (E
-value < 0.001; ). When compared with the environmental sequence database (ENV), 60% of DsH and 77% of DsB sequences had significant hits (E
-value < 0.001; ). When NR and ENV hits were compiled, 35% of DsH and 18% of DsB sequences were entirely novel (i.e. they had no hits to either database). This percentage of novel sequences is lower than that observed in other viral metagenomes (Breitbart et al., 2002
; Angly et al., 2006
), but there still remains a large fraction of coral-associated viruses with no significant similarity to any known sequences.
Summary of tblastx hits to GenBank NR and ENV databases.
Animal viruses associated with corals
Remarkably, 4.3% of all DsH sequences and 7.6% of all DsB sequences had significant hits to herpesviruses when compared with the NR database (). This represents 69% and 84% of the hits to eukaryote-specific viruses in the DsH and DsB libraries respectively. The most common herpesvirus hits were cercopithecine herpesvirus 2 sequences in the DsH library and cercopithecine herpesvirus 1 sequences in the DsB library (summarized in ; listed in full in Table S2). These are both alphaherpesviruses, but significant hits to all subfamilies of the family Herpesviridae were observed.
Summary of tblastx hits to GenBank NR database.
Two features of the tblastx
hits indicate that the coral viral community did not contain known herpesviruses, but rather ‘herpes-like’ viruses. First, sequence similarity was rarely above 70% amino acid identity. Second, many sequences hit simple repeats in complete herpesvirus genomes. These repeats are characteristic of herpesviruses (McGeoch et al., 2006
), but not diagnostic given the level of sequence divergence. To better visualize these herpes-like sequences, we used the location of the best tblastx
hit for each sequence to plot sequence hit along the most commonly identified herpesvirus genomes (). These hits were distributed fairly evenly across the target genomes, indicating that there are many regions of similarity. This supports the identification of these viruses as ‘herpes-like’.
Fig. 2 Alignment of coral virus sequences to alphaherpesvirus genomes. Each dot represents the location of an individual metagenome sequence based on its best tblastx hit (E-value < 0.001) to the virus genome. Shown are the five alphaherpesviruses with (more ...)
To better characterize sequences functionally, DsH and DsB were compared with a database of complete, annotated genomes from eukaryote-specific viruses using blastx (E-value < 0.001; accession numbers of genomes included in the database are listed in Appendix S1). Eight sequences from each metagenome had a significant hit to a herpesvirus gene (Table S3). This included a total of four hits to genes involved in nucleotide metabolism (e.g. ribonucleoside-phosphate reductase), four hits to predicted or known glycoprotein genes, and four hits to genes for latency-associated nuclear antigens. The small number of hits demonstrates the high degree of sequence novelty in the coral-associated viral communities and further supports the notion that the ‘herpes-like’ viruses associated with corals are highly divergent from any previously studied viruses. Nevertheless, the tblastx and blastx results together show that a subset of coral-associated viruses is more similar to herpesviruses than to anything else known.
Herpes-like sequences did not comprise a large percentage of hits in previously published viral metagenomes from nearshore seawater, global ocean seawater, marine sediment or human feces (Breitbart et al., 2002
; Angly et al., 2006
). The occurrence of these sequences at such high abundance is therefore novel to corals. This should expand the scope of research on coral disease, but more work is needed before the potential pathogenicity of these viruses is known.
Algae and plant viruses associated with corals
Hits to viruses that infect algae and plants (family Phycodnaviridae) were also observed in the tblastx comparison to the NR database (summarized in , listed in full in Table S2). The DsH library contained one hit to Ectocarpus siliculosus virus, a virus of brown algae. The DsH and DsB libraries included three and four hits, respectively, to chlorella viruses, which infect green algae. Each library also contained four hits to Emiliania huxleyi virus 86, a virus of coccolithophores. The presence of phycodnavirus genes in the ‘bleaching’ DsB sequences is consistent with the fact that this metagenome was created from corals that had only partially bleached when they were collected. This tissue therefore still contained large numbers of symbiotic algae, which could serve as targets for phycodnaviruses.
When compared with the database of virus genomes using blastx
, 30 DsH sequences and 22 DsB sequences had significant hits to phycodnavirus genes (Table S4; E
-value < 0.001). Nearly all of these genes coded for hypothetical or putative proteins. Most functional predictions, when available, involved nucleotide metabolism (e.g. thymidylate synthase and ribonucleoside-triphosphate reductase). Although the phycodnavirus hits comprised fewer than 10% of hits to eukaryote-specific viruses in both tblastx
searches, their presence is notable because some phycodnaviruses are known to infect the symbiotic microalgae of hydra (Van Etten et al., 1982
). This collection of hits suggests that a subset of coral-associated viruses may target algal cells in the coral holobiont. Thus, the potential remains for coral viruses to contribute to coral bleaching by directly infecting zooxanthellae, as researchers have previously posited (Wilson et al., 2005
When compared with the NR database with tblastx, 29% of the phage hits in DsH and 44% of the phage hits in DsB were to cyanophage sequences (summarized in ; full list in Table S2). These phages represent a guild that is known to infect cyanobacteria rather than a taxonomic group of phages. For example, cyanophages P-SSP7 and P60 are podoviruses, but cyanophages P-SSM2 and P-SSM4 are myoviruses. DsH and DsB both contained sequences with high similarity to all four of these cyanophages.
To further characterize the phage hits, DsH and DsB were compared with a database containing only phage genes (the Phage Sequence Databank, http://phage.sdsu.edu/phage
) using blastx
-value < 0.001). A total of 173 DsH sequences and 153 DsB sequences had significant hits to cyanophage genes. These hits represented nearly all functional categories of cyanophage genes, including tail and capsid components, nucleotide metabolism, DNA replication, DNA repair and protein translation (Table S5). Interestingly, there were extremely strong hits in both libraries to the core photosystem II reaction centre protein encoded by the psbA
gene. To ensure that these photosystem hits were not derived from coral zooxanthellae symbionts (Symbiodinium
spp.), which also have psbA
genes, each sequence was compared with the GenBank NR database using blastn
-value < 0.001). The NR database contains psbA
genes from four different Symbiondinium
clades; however, there were no significant hits to these sequences in any of the comparisons. The core photosystem II reaction centre proteins encoded by the psbA
gene were recently shown to be present and functional in cyanophage genomes (Sullivan et al., 2006
). Their presence here lends supports to the identification of these coral-associated viruses as cyanophages.
The discovery of cyanophages in the coral viral assemblage is not unexpected. Cyanobacteria were previously identified in one of four 16S rDNA clone libraries from D. strigosa
(Rohwer et al., 2002
) and as endosymbionts in the Caribbean coral Montastraea cavernosa
(Lesser et al., 2004
). The cyanobacteria in M. cavernosa
belong to the Order Chroococcales
. This Order also contains Prochlorococcus
spp. and Synechococcus
spp., which are the targets of the cyanophages identified in the DsH and DsB libraries.
It has been proposed that cyanobacterial symbionts of corals perform nitrogen fixation within either the coral skeleton or tissue (Shashar et al., 1994
; Lesser et al., 2004
). Nitrogen fixation within the tissue has now been demonstrated in M. cavernosa
(Lesser et al., 2007
). Zooxanthellae are typically thought to be nitrogen limited, but the authors of this study showed that zooxanthellae could acquire fixed nitrogen directly from endosymbiotic cyanobacteria. Thus, the infection of cyanobacteria by cyanophages could determine the ability of zooxanthellae to acquire fixed nitrogen within the coral holobiont.
Vibriophages made up 3.7% and 6.0% of DsH and DsB phage hits, respectively, to the NR database (summary in , full list in Table S2). When compared with the Phage Sequence Databank using blastx, DsH and DsB contained 67 and 80 hits, respectively, to vibriophage genes, including those involved in tail construction, protein translation, nucleotide metabolism, and DNA packaging, replication and repair (Table S6).
A subset of Vibrio
spp. known to cause coral bleaching and disease has become the basis for model systems used to examine the interactive effects of microbes and temperature on coral physiology (Kushmaro et al., 2001
; Ben-Haim et al., 2003
). Recently, phage therapy with cultured vibriophages was shown to prevent tissue necrosis and bleaching in Pocillopora damicornis
specimens experimentally infected with the bacterium Vibrio coralliilyticus
(Efrony et al. 2007
). Vibriophages in the coral holobiont could similarly infect bacterial pathogens and therefore benefit the coral; however, their presence could be detrimental if they disrupt the coral holobiont (e.g. by releasing toxins or clearing space for more virulent strains). Further investigations should be conducted to determine the degree of influence that vibriophages have on their target populations in the complex environment of the coral holobiont.
The abundance of cyanophages and vibriophages in these sequence libraries should not be taken to represent precise phage abundances in nature. These two groups are well studied and well represented in GenBank. As other microbial symbionts of corals are identified and their phages are studied, the relative abundances of cyanophages and vibriophages identified in coral viral metagenomes will decrease. The functional capacities of these two groups, however, will remain an important focus of attention.
Statistical comparison of phage communities
UniFrac (Lozupone et al., 2006
), a metric of the unique phylogenetic distance between communities (Lozupone and Knight, 2005
), was used to compare the phage populations in our coral virus metagenomes to each other and to two previously obtained phage sequence libraries: Reef (from water collected at four coral reefs) and Ocean (a pooling of sequences from four oceanic provinces; Angly et al., 2006
). There was no significant difference between the DsB and DsH samples in this analysis (P
= 0.94). A clustering of environments based on the UniFrac metric showed that these communities were more similar to each other than to the Ocean or Reef communities, with 100% jackknife support for all nodes. Because this method compares metagenome sequences to a phylogeny of known phages, it cannot reveal differences in phages that are undescribed. Because we chose to pool tissue samples prior to DNA extraction, this method also cannot reveal variability between individuals. In the context of known phage families, the coral phage communities do not have significant differences from each other, however, they do cluster together when compared with other marine phage communities, which suggests that the coral holobiont is a distinct environment from that of the surrounding seawater. This falls in line with previous studies demonstrating that coral bacterial communities differ from those in the surrounding seawater (Rohwer et al., 2001b
; Frias-Lopez et al., 2002
; Ritchie, 2006
Viral community structure
To characterize the sequence diversity of coral-associated viral communities as a whole, metagenome sequences were assembled into groups of contiguous sequences (contigs). The number of sequences in each contig was tallied and the frequencies of contigs of each size were used to predict characteristics of the viral communities (Breitbart et al., 2002
; Angly et al., 2005
) using PHACCS (Angly et al., 2005
). The DsH metagenome contained 1523 singletons, 22 contigs of two sequences, three contigs of three sequences and one contig of four sequences (thus, the contig spectrum was [1523, 22, 3, 1]; Table S7). In the DsB metagenome, contig construction yielded only singletons and two-sequence contigs, which are not considered sufficient for accurate modelling. Based on its contig spectrum, the DsH community was predicted to have a total of 28 600 viral types and a Shannon–Weiner index of 8.96. The most dominant genotype was predicted to comprise 2.6% of the total viral community. This is extraordinary diversity, comparable to that of viral communities in soil (Fierer et al., 2007
) and more diverse than viral communities in seawater (Breitbart et al., 2002
Implications for the coral holobiont
Interest in coral microbiology has surged with the recognition that coral-associated microbes may serve as pathogens or as mutualists that perform nitrogen fixation, vitamin and nutrient scavenging, antimicrobial production or space filling. The relative importance of each potential role has yet to be elucidated; however, bacteriophages should be expected to affect these microbial communities and their functionality. Phages are responsible for 50% of bacterial death in the ocean (Wilcox and Fuhrman, 1994
) but may be responsible for a much higher percentage in the coral holobiont if the mobility of protist predators is restricted by the coral's tissue and mucus. Thus, the differential infection of bacterial groups by phages might serve as a ‘top-down’ control on the diversity of the coral-associated microbial consortium and its ability to fill the various roles described above.
The coral animal itself is also expected to regulate its microbial communities. It was demonstrated that sterilized coral mucus acts as a selective agent, promoting the growth of non-pathogenic and antibiotic-producing bacterial strains over known pathogens; this selectivity was not observed when mucus was collected during a bleaching event (Ritchie, 2006
). Changes in coral mucus chemistry over long (evolutionary) or short (ecological) timescales can therefore provide ‘bottom-up’ control over a community of microbial associates. The relative extent to which a coral or its phage population is able to exert such control on a microbial community is not yet known, but this study shows that further research is needed to understand the role of phages in structuring these communities.
Microbial abundances in coral tissues are approximately 107
(Wegley et al., 2004
). In most environments, there are 10 VLPs for every microbial cell, the majority of which are phage. If this held true for the coral holobiont microenvironment, we would expect corals to have 108
VLPs per cm2
. Considering the large number of hits to eukaryote- and algae-specific viruses in our metagenomic libraries, the typical viral abundance on corals might be much greater. While the degree of natural variability in coral virus abundance remains to be determined, we propose that observations of extraordinary numbers of viruses in apparently healthy corals may represent a diversity of functions rather than a severity of infection.
Studies of coral-associated viruses often describe these communities as a latent pathogen reservoir, susceptible to induction by environmental stressors. The presence of eukaryote-specific viruses is demonstrated by the sequence data presented here, and it is indeed likely that some coral pathologies are caused by viral vectors. However, it appears that corals are chronically infected by thousands of viral strains. The DNA used to create our metagenomes was extracted from viral particles, thus these sequence libraries represent not latent viral sequence embedded within eukaryote genomes but viral particles present in the coral tissue at the time of collection. The presence alone of viruses is therefore not indicative of disease.
Furthermore, the observed genetic diversity of viruses in the coral holobiont casts the very nature of coral symbiosis in a new light. Lesser and colleagues (2007
) showed that while coral-associated cyanobacteria could provide fixed nitrogen to zooxanthellae, zooxanthellae did not appear to depend on this source of nitrogen. Therefore, the mechanism maintaining coral–zooxanthellae symbiosis was not fully understood. Villarreal (2007
) has suggested that viruses can serve as a stabilizing force for symbioses by establishing addiction systems within a host. For example, zooxanthellae infected with a latent virus are resistant to lysis by VLPs (Wilson and Chapman, 2001
). Phages may also stabilize symbioses. For example, a phage specific to Hamiltonella defensa
, a bacterial symbiont of the pea aphid, produces a toxin that appears to protect host aphids from eukaryotic parasites (Moran et al., 2005
). The diversity of constituents in the coral holobiont elevates the potential for these types of viral functions to exist therein. In fact, the stability of the holobiont itself may ultimately depend on the action of viruses. The study of viruses within the coral holobiont will shed new light on the basic biology of symbiosis, but it will also be particularly important as corals face ever-increasing threats to their health and habitats.
Here we have described the complexity of an under-studied facet of the coral holobiont. Herpes-like viruses occur in both healthy and bleaching corals. This should be a focus for future research on coral holobiont complexity, symbiosis and immunology. The largest identified functional group of coral-associated viruses, cyanophages, may affect the population structure of symbiotic cyanobacteria and endolitic algae, while vibriophages present in coral tissue may affect the pathogenesis of coral-associated Vibrio spp. While these are important structuring forces for the coral holobiont, the prediction that up to 28 600 viral types occur in a healthy coral's viral community indicates that there are myriad functions and interactions still unidentified in this viral assemblage. When compared in the framework of a phage phylogenetic tree, coral-associated phage communities from bleaching and healthy corals are not significantly different from each other, but the coral holobiont as a phage environment is distinct from that of coral reef and oceanic waters. Thus, it appears that a diverse community of viruses continuously occupies coral tissues. With the potential to target animal, algal and microbial cells, viruses are likely to be crucial in maintaining the overall function of the coral holobiont.